The invention relates in general to the diagnosis and treatment of immune disorders.
In the cytosol, there is a soluble proteolytic pathway that requires ATP and involves covalent conjugation of the cellular proteins with the small polypeptide ubiquitin, or Ub, (Hershko et al., 1992, Ann. Rev. Biochem., 61: 761-807; Rechsteiner et al., 1987, Ann. Rev. Cell. Biol., 3: 1-30). Therafer, the conjugated proteins are hydrolyzed by a 26S proteolytic complex containing a 20S degradative particle called the proteasome (Goldberg, 1992, Eur. J. Biochem., 203: 9-23); Goldberg et al., 1992, Nature, 357: 375-379). This multicomponent system is known to catalyze the selective degradation of highly abnormal proteins and short-lived regulatory proteins. However, the system also appears to be responsible for the breakdown of most proteins in maturing reticulocytes (Boches et al., 1982, Science, 215: 978-980); Spenser et al., 1985, J. Biol. Chem., 257: 14122-14127), in growing fibroblasts (Ciechanover et al., 1984, Cell, 37: 57-66; Gronostajski et al., 1985, J. Biol. Chem, 260: 3344-3349) and in atrophying skeletal muscle.
The first step in degradation of many proteins involves their conjugation to Ub by an ATP-requiring process, as described below. The ubiquitinated proteins are then degraded by an ATP-dependent proteolytic complex, referred to above, known as the 26S proteasome complex.
The precise nature of the 26S proteasome complex is unclear, although it has been shown that the 1060-1500 kDa (26S) complex can be formed in extracts of energy-depleted reticulocytes by an ATP-dependent association of three components, refereed to as OF-1, OF-2, and OF-3 (Ganoth et al., 1988, J. Biol. Chem., 263; 12412-12419). A large (xcx9c700 kDa) multimeric protease found in the cytoplasm and nucleus of eukaryotic cells, referred to as the proteasome, is a component (OF-3) (Driscoll et al., 1992, J. Biol. Chem., 265: 4789-4792; Eytan et al., 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 7751-5 7755; Orlowski et al., 1990, Biochemistry, 29: 10289-10297; Rivet, 1989, Arch. Biochem. Biophys., 268: 1-8). 
The proteasome is believed to make up the catalytic core of the large 26S multisubunit cytoplasmic particle necessary for the ubiquitin-dependent pathway of intracellular proteolysis (Driscoll et al., 1990, J. Biol., Chem., 265: 4789-4692; Eytan et al., 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 7751-7755; Hough et al., 1987, Biochemistry, 262: 8303-8313; McGuire et al., 1988, Biochim. Biophys. Acta., 967: 195-203; Rechsteiner et al., 1987, Ann. Rev. Cell. Biol., 3: 1-30; Waxman et al., 1987, J. Biol. Chem., 262: 2451-2457). By itself, the proteasome is unable to degrade ubiquitinated proteins, but provides most of the proteolytic activity of the 26S proteasome complex.
There is another ATP-dependent protease that is involved in degradation of ubiquitinated proteins, forms a complex with the proteasome and appears to be part of the 26S proteasome complex, which rapidly degrades proteins conjugated to ubiquitin. This protease, referred to as multipain, has been identified in muscle and plays an essential role in the ATP/ubiquitin-dependent pathway.
The complex formed between multipain and proteasome in vitro appears very similar or identical to the 1500 kDa Ub-conjugate, degrading enzyme, or 26S proteolytic complex, isolated from reticulocytes and muscle. The complexes contain the characteristic 20-30 kDa proteasome subunits, plus a number of larger subunits, including the six large polypeptides found in multipain. The complex formed contains at least 10-12 polypeptides of 40-150 kDa. A 40 kDa polypeptide regulator of the proteasome, which inhibits the proteasome""s proteolytic activities has been purified from reticulocytes and shown to be an ATP-binding protein whose release appears to activate proteolysis. The isolated regulator exists as a 250 kDa multimer and is quite labile (at 42xc2x0 C.). It can be stabilized by the addition of ATP or a nonhydrolyzable ATP analog, although the purified regulator does not require ATP to inhibit proteasome function and lacks ATPase activity. The regulator has been shown to correspond to an essential component of the 1500 kDa proteolytic complex. The regulator appears identical to CF-2 by many criteria. These findings suggest that the regulator plays a role in the ATPxe2x80x94dependent mechanism of the 26S proteasome complex.
The 20S proteasome is composed of about 15 distinct 20-30 kDa subunits. It contains at least three different peptidases that cleave specifically an the carboxyl side of the hydrophobic, basic, and acidic amino acids (Goldberg et al., 1992, Nature, 357: 375-379: Goldberg, 1992, Eur. J. Biochem., 203: 9-23; Orlowski, 1990, Biochemitry, 29: 10289-10297; Rivett et al., 1989, Arch. Biochem. Biophys., 218: 1; Rivett et al., 1989, J. Biol. Chem., 264: 12215-12219;Tanaka et al., 1992, New Biol. 4: 1-11). These peptidases are referred to as the chymotrypsin-like peptidase, the trypsin-like peptidase, and the peptidylglutamyl peptidase. Which subunits are responsible for these activities is unknown although the cDNA""s encoding several subunits have been cloned (Tanaka et al., 1992, New Biol., 4: 1-11).
As reviewed by Hopkin (1997, J. NIH Research, 9: 36-42) and briefly summarized herein, insight into the mechanisms by which proteolysis is controlled come from studies of the eukaryotic cell cycle. To proceed through the cell cycle, replicating its genome and dividing the resulting DNA between daughter cells during mitosis, a cell must appropriately activate and inactivate the regulators of cell division, the cyclin-dependent kinases (Cdks). To control Cdks, cells can specifically degrade the cyclin proteins that activate Cdks and the inhibitors that inactivate them. One mechanism by which specificity in targeted proteolysis is achieved is ubiquitination, the process by which cells tack long chains of a 76-amino acid marker protein called ubiquitin (Ub) onto proteins that are destined for destruction. Ubiquitination of a handful of cyclins and Cdk inhibitors leads to their timely demise and allows a cell to complete mitosis or to replicate its DNA; further, it is believed that phosphorylation of unstable proteins, such as the cyclins, often increases their susceptibility to ubiquitination and subsequent elimination.
As described below, ubiquitination affects signal transduction, as it may mark certain cell-surface growth-factor receptors for endocytosis and destruction; further, it is known that ubiquitination, coupled with phosphorylation, stimulates the signaling pathway that activates the transcription factor NFxcexaB. Ubiquitin also plays a role in protein degradation pathways regulating cell differentiation and death during development
Ubiquitination and the Cell Cycle
Evidence that ubiquitination was interesting from the point of view of regulation came with the development of a mouse cell line that arrests in the G2, or gap 2, phase of the cell cycle; these cells harbor a defect that cripples an enzyme that activates Ub before it can bind to proteins, such as the cyclins, that must be targeted for destruction. Prior to this work, ubiquitination was viewed only as a means for eliminating damaged, denatured, and misfolded proteins.
Most of the proteolysis that occurs in cells involves the degradation of Ub-conjugated proteins. As stated above, the proteasome recognizes the polyubiquitin tag, selectively admits proteins to which this marker is complexed and then cleaves them into small peptide fragments. Ubiquitination is dependent upon a series of proteins named for their order of elution from a Ub-affinity column. Ub-activating enzymes, called Els, prime Ub for transfer to a substrate protein by forming a temporary thioester linkage between a terminal glycine of Ub and one of their own cysteine residues. Enter the Ub-conjugating proteins generically called E2s. These enzymes accept activated Ub from an E1 and transfer it to the substrate protein, either directly or with the help of a Ub-ligase protein, or E3; interactions between different E2s and E3s may contribute to the substrate specificity of the ubiquitination reaction. Yeast maintain a cadre of more than a dozen structurally related E2s as well as a handful of E3s (reviewed by Haas and Siepmann, 1997, FASEB J., 11: 1257-1268). Functional homologues of these proteins have been found in humans (see Honda et al., 1997, FEBS Lett., 420: 25-27).
Even within the cell cycle, different sets of E2s and E3s function to mark cyclins and Cdk inhibitors for destruction. Together these proteins regulate entry into new cycles of cell division, initiation of DNA replication, and the onset of mitosis. In yeast, cyclins bind to and activate Cdc28, which then pushes cells into the next phase of the cell cycle, initiating cell division. It is said that the concentrations of both the cyclins and the Cdk inhibitors that drive the cell cycle through their interactions with Cdc28 may be tightly controlled by Ub-associated proteolysis. The G1 cyclins Cln1, Cln2, and Cln3 activate Cdc28, by which they are then reciprocally phosphorylated; this phosphorylation marks the cyclins for ubiquitination and subsequent destruction by the proteasome.
The Ub-ligase complex that ubiquitinates the cell-cycle proteins that control the completion of mitosis is known to be activated by phosphorylation. The coupling of cyclin B and its kinase Cdc2 initiates mitosis in yeast. In that system, cyclin B accumulates during interphase until its pairing with Cdc2 drives the cell into mitosis and leads to its eventual destruction. The cyclosome (also called the anaphase-promoting complex , or APC), a 20S nuclear particle which serves as the Ub-ligase complex, helps to ubiquitinate the mitotic cyclins A and B as well as the as-yet-unidentified xe2x80x9cgluexe2x80x9d proteins that bind sister chromatids together during metaphase. Late in mitosis, an unknown kinase phosphorylates and activates the cyclosome/APC. Then, working in conjunction with a Ub-conjugating enzyme called E2-C in clams (an organism favored by cell-cycle researchers), the cyclosome marks the mitotic cyclins for degradation by the proteasome (Aristarkhov et al., 1996, Proc. Nat. Acad. Sci. U.S.A., 93: 9303-9307); Ub-directed destruction of the mitotic cyclins leads to the inactivation of Cdc2 and the degradation of the xe2x80x98gluexe2x80x9d proteins, so that sister chromatids are allowed to segregate into the two daughter cells. E2-C and its human homologue, the ubiquitin-conjugating human enzyme UbcH10, have been characterized in detail (Townsley et al., 1997, Proc. Natl. Acad. Sci. U.S.A., 94: 2362-2367).
ii Cell Signalling Pathways
Proteins that control cell-cycle progression may respond to environmental cues, such as are provided by growth factors. Growth factor-stimulated signaling pathways are, themselves controlled in part by ubiquitination. One of the best studied examples is the NFxcexaB pathway (see below). Binding of the cytokine tumor necrosis factor-xcex1(TNF-xcex1) to cell-surface receptors, or the occurrence of another proinflammatory or stress event (e.g. hypoxia), initiates a signaling cascade that activates NFxcexaB (see below) and c-Jun, transcription factors that govern the proliferative response in cells.
Ubiquination may be involved in regulating the amount of a receptor present on the cell membrane. Stimulation of the Met tyrosine-kinase receptor by the ligand hepato-cyte growth factor/scatter factor (HGF/SFD spurs the embryonic development of a variety of mammalian tissues, including liver, placenta, and muscles). For example, it has been reported that HGF/SF stimulates the degradation of the Met tyrosine-kinase receptor by proteasomes in a human sarcoma cell line (Jeffers et al., 1997, Mol. Cell. Biol., 17: 799-808). In the absence of HGF/SF, this receptor is cleaved by an unknown protease and the fragment containing the tyrosine-kinase activity remains embedded in the cell membrane. According to Hopkin et al. (1997, supra), it has been postulated that the presence of an unregulated tyrosine kinase in the membrane could be dangerous and that Ub-targeted degradation is intended to rid the cell of the membrane-embedded kinase fragment before damage can occur.
It is thought that the proteasome will cleave any ubiquitinated protein with which it comes in contact; however, different receptors may recognize substrates bearing Ub chains that differ in internal. The 2-megadalton proteasome complex, which comprises four stacked rings of xcex1 and xcex2 protein subunits with a series of protease-active sites lining the inside of the resulting tube, recognizes a subset of ubiquitin chains via the S5 protein subunit. After a Ub-tagged protein binds to the proteasome complex, it is unfolded in order to facilitate passage through the proteasome pore into the proteolytic chamber. Mutational inactivation of the S5 proteasome subunit results in a specific subset of ubiquitinated proteins being spared from degradation (van Nocker et al., 1996, Mol. Cell. Biol., 16: 6020-6028). It is this selectivity which suggests that the proteasome may possess more than one receptor for detecting Ub-conjugated proteins.
NFxcexaB has been implicated in the etiology of immune disorders. Adams et al. (WO 96/13266) teach inhibition of proteasome activity, which mediates the activation of NFxcexaB, to treat autoimmune diseases.
Similarly, Brand et al. (WO 95/24914) teach that new, as well as existing, proteasome inhibitors may be used to treat autoimmune diseases.
Further, according to Palombella et al. (WO 95/25533; page 7, lines 16-23), Goldberg et al. are said to teach methods and drugs that inhibit antigen processing for the treatment of autoimmune diseases.
According to Kopp and Ghosh (1994, Science, 265: 956-969) and Grilli et al. (1996, Science, 274: 1383-1385), salicylate and glucocorticoids, anti-inflammatory drugs that are inhibitors of NFxcexaB, are widely used to treat established cases of autoimmune diseases.
In addition, NFxcexaB is said to said to be a positive transcriptional regulator of inducible nitric oxide synthase (iNOS), which in turn mediates cytokine-induced inhibition of insulin secretion by pancreatic cells of the islets of Langerhans (Kwon et al., 1995, Endocrinonlogy, 136: 4790-4795); inhibition of NFxcexaB activity suppresses this phenotype.
There is need in the art for improved methods of treating autoimmune disorders.
The invention provides a method of detecting autoimmune disease in a mammal, comprising providing a biological sample from a mammal and detecting proteasome activity, wherein a reduction in proteasome activity from a basal state is indicative of autoimmune disease.
As used herein, the term xe2x80x9cautoimmune diseasexe2x80x9d refers to a disorder wherein the immune system of a mammal mounts a humoral or cellular immune response to the mammal""s own tissue or has intrinsic abnormalities in its tissues preventing proper cell survival without inflammation.
Examples of autoimmune diseases include, but are not limited to, diabetes, rheumatoid arthritis, multiple sclerosis, lupus erythematosis, myasthenia gravis, sclerodenna, Crohn""s disease, ulcerative colitis, Hashimoto""s disease, Graves"" disease, Sjxc3x6gren""s syndrome, polyendocrine failure, vitiligo, peripheral neuropathy, graft-versus-host disease, autoimmune polyglandular syndrome type I, acute glomerulonephritis, Addison""s disease, adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis, amyotrophic lateral sclerosis, ankylosing spondylitis, autoimmune aplastic anemia, autoimmune hemolytic anemia, Behcet""s disease, Celiac disease, chronic active hepatitis, CREST syndrome, dermatomyositis, dilated cardiomyopathy, eosinophilia-myalgia syndrome, epidermolisis bullosa acquisita (EBA), giant cell arteritis, Goodpasture""s syndrome, Guillain-Barrxc3xa9 syndrome, hemochromatosis, Henoch-Schxc3x6nlein purpura, idiopathic IgA nephropathy, insulin-dependent diabetes mellitus (IDDM), juvenile rheumatoid arthritis, Lambert-Eaton syndrome, linear IgA dermatosis, myocarditis, narcolepsy, necrotizing vasculitis, neonatal lupus syndrome (NLE), nephrotic syndrome, pemphigoid, pemphigus, polymyositis, primary sclerosing cholangitis, psoriasis, rapidly-progressive glomerulonephritis (RPGN), Reiter""s syndrome, stiff-man syndrome and thyroiditis.
As used herein, the term xe2x80x9cdiabetesxe2x80x9d refers both to the type I form of the disease and to type II cases that share only an islet cell defect with type I.
Symptoms common to many types of autoimmune dysfunction include, but are not limited to: fatigue; inflammation; paresis; joint stiffness, pain or swelling; skin lesions or nodules; skin discoloration; enzymatic imbalances; tissue degeneration. Examples of such symptoms as pertain to specific autoimmune diseases are described hereinbelow in the Description section. Such symptoms or, alternatively, measurements of tissue death/destruction, may be used either as diagnostic indicators of the presence of an autoimmune disease, or as indices by which to assess the efficacy of treatment thereof.
In the treatment of autoimmune disease, a therapeutically effective dosage regimen should be used. By xe2x80x9ctherapeutically effectivexe2x80x9d, one refers to a treatment regimen sufficient to restore the the mammal to the basal state, as defined herein, at the cellular or tissue site of manifestation or to prevent an autoimmune disease in an individual at risk thereof or restore the mammal""s immune system to the basal state. Alternatively, a xe2x80x9ctherapeutically effective regimenxe2x80x9d may be sufficient to arrest or otherwise ameliorate symptoms of an autoimmune disease. Generally, in the treatment of autoimmune diseases, an effective dosage regimen requires providing the medication over a period of time to achieve noticeable therapeutic effects; such a period of time may begin at, or even before, birth and continue throughout the life of the individual being treated. Methods of treatment are discussed in detail in the Description section, below.
As used herein, the term xe2x80x9cbiological samplexe2x80x9d refers to a whole organism or a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). xe2x80x9cBiological samplexe2x80x9d further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof Lastly, xe2x80x9cbiological samplexe2x80x9d refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules.
As used herein, the term xe2x80x9corganismxe2x80x9d refers to all cellular life-forms, such as prokaryotes and eukaryotes, as well as non-cellular, nucleic acid-containing entities, such as bacteriophage and viruses.
As used herein, the term xe2x80x9cmammalxe2x80x9d refers to a member of the class Mammalia, including a human.
It is contemplated that procedures useful for the detection of proteins or nucleic acids and biological activities thereof include, but are not limited to, immunological assays, such as immunoblotting, immocytochemistry, immunohistochemistry or antibody-affinity chromatography, electrophoretic analysis, such as one- or two-dimensional SDS-PAGE, Northern or Southern analysis, in vivo or in vitro enzymatic activity assay, the polymerase chain reaction (PCR), reverse-transcription PCR (RT-PCR), in situ nucleic acid hybridization, electrophoretic mobility shift analysis (EMSA), transcription assay, or variations or combinations of these or other techniques such as are known in the art.
As used herein, the term xe2x80x9cproteasomexe2x80x9d refers to a multi-subunit protein complex in the cytoplasm of eukaryotic cells which recognizes and selectively cleaves ubiquitinated protein molecules to mediate either activation or degradation of the protein so recognized and cleaved.
As used herein in reference to proteasome activity, the term xe2x80x9creductionxe2x80x9d refers to the failure of the proteasome to cleave a target ubiquitinated protein at as few as one-, more than one-, or even as many as all of the sites that it normally (ie., in a genetically wild-type or otherwise healthy individual) recognizes and cleaves in that protein. Preferably, such a reduction involves failure to cleave the target protein at 5-10% of sites, more preferably, at 20-50% of sites, and most preferably at 75-100% of such sites. Different numbers and/or patterns of sites on different proteins are cleaved by the proteasome. The term xe2x80x9cdifferent proteinsxe2x80x9d refers to protein molecules that differ in amino acid sequence in at least one position. Promiscuous cleavage (i.e., at a site not normally recognized and cleaved) of a protein by the proteasome is defined as a reduction only if such aberrant cleavage is accompanied by the failure of the proteasome to cleave a site normally recognized and cleaved.
As used herein, the term xe2x80x9cbasal statexe2x80x9d refers to the level of activity of a protein, nucleic acid or other molecule where autoimmune disease is not present, i.e. a xe2x80x9cnormal levelxe2x80x9d of activity. The basal state is observed in genetically wild-type or otherwise healthy individuals, as well as in individuals who have a propensity for an autoimmune disease (as judged by genetic or environmental criteria known to those of skill in the medical art) but have not yet developed such a disease and even individuals who are in the early stages of an autoimmune disease but have not, for example, become actively symptomatic.
Preferably, the biological sample comprises protein.
It is contemplated that the protein of a biological sample of use in the invention may be crude (i.e., in an unfractionated cell lysate), partially-purified or isolated, and either naturally-occurring or produced by recombinant techniques, such as the expression of a cDNA or other gene sequence cloned from a mammal.
In a preferred embodiment, a reduction in proteasome activity is detected.
A reduction in proteasome activity may be observed as a reduction in the activation of transcription factors (among them, NFxcexaB) as judged either by observation of the physical properties of such a protein (for example, antigenicity or molecular weight, as judged by sedimentation or electrophoretic mobility) that are characteristic of its pre-activation form or by the absence of mRNA (or the protein encoded by such a message) resulting from the transcription of a gene that is positively regulated by the protein in a biological sample. In addition, a reduction in the proteolytic processing of a protein normally cleaved by the proteasome (such as an MHC antigen, which is cleaved by the proteasome prior to transport to- and presentation on the cell surface).
Preferably, the reduction in proteasome activity comprises a reduction of proteolytic processing of NFxcexaB, p105, p100, IxcexaB, or a subunit thereof
Methods for the detection of a reduction in proteolytic processing of NFxcexaB are as described in detail hereinbelow in Example 2.
Preferably, the mammal is a human.
It is preferred that the autoimmune disease is an HLA class II-linked disease.
As used herein, the term xe2x80x9cHLA class II-diseasexe2x80x9d refers to those autoimune diseases showing statistical risk factors for disease penetrance attributed to HLA class II genes or to neighboring genes.
The term xe2x80x9cHLAxe2x80x9d (for xe2x80x9chuman lymphocyte antigenxe2x80x9d) refers to genes of the human major histocompatibility complex (MHC) or their protein products. In mice, the genetic region corresponding to or homologous with the HLA is termed the H2 complex.
In another preferred embodiment, the autoimmune disease is selected from the group that includes those diseases listed above as autoimmune diseases.
Another aspect of the present invention is a method of detecting autoimmune disease in a mammal, comprising providing a biological sample from a mammal and detecting protein ubiquitination, wherein a reduction in protein ubiquitination from a basal state is indicative of autoimmune disease.
As used herein in reference to protein ubiquitination, the term xe2x80x9creductionxe2x80x9d refers to the failure of ubiquitinating enzymes to ubiquitinate a target protein at as few as one-, more than one-, or even as many as all of the sites that they normally (ie., in a genetically wild-type or otherwise healthy individual) recognize and ubiquitinate in that protein. Preferably, such a reduction involves failure to ubiquitinate the target protein at 10-20% of sites, more preferably, at 40-50% of sites, and most preferably at 80-100% of sites. Different numbers and/or patterns of sites on different proteins are ubiquitinated by the ubiquitinating enzymes. The term xe2x80x9cdifferent proteinsxe2x80x9d refers to protein molecules that differ in amino acid sequence in at least one position. Promiscuous ubiquitination (i.e., at a site not normally recognized and ubiquitinated) of a protein by the ubiquitinating enzymes is defined as a reduction only if such aberrant ubiquitination is accompanied by the failure of the ubiquitinating enzymes to ubiquitinate a site normally recognized and ubiquitinated.
It is preferred that the biological sample comprises protein.
It is additionally preferred that a reduction in protein ubiquitination is detected for a protein.
Preferably, the mammal is a human.
It is preferred that the autoimmune disease is an HLA class II-linked disease.
In another preferred embodiment, the autoimmune disease is selected from the group that includes those diseases listed above.
The invention also encompasses a method of detecting autoimmune disease in a mammal, comprising providing a biological sample from a mammal and detecting protein phosphorylation, wherein a reduction in protein phosphorylation from a basal state is indicative of autoimmune disease.
As used herein in reference to protein phosphorylation, the term xe2x80x9creductionxe2x80x9d refers to the failure of a protein kinase to phosphorylate a target protein at as few as one-, more than one-, or even as many as all of the sites that it normally (ie., in a genetically wild-type or otherwise healthy individual) recognizes and phosphorylates in that protein. Preferably, such a reduction involves failure to phosphorylate the target protein at 2-10% of sites, more preferably, at 25-50% of sites, and most preferably at 90-100% of sites. Different numbers and/or patterns of sites on different proteins are phosphorylated by protein kinases. The term xe2x80x9cdifferent proteinsxe2x80x9d refers to protein molecules that differ in amino acid sequence in at least one position. Promiscuous phosphorylation (i.e., at a site not normally recognized and phosphorylated) of a protein by a protein kinase is defined as a reduction only if such aberrant phosphorylation is accompanied by the failure of the protein kinase to phosphorylate a site normally recognized and phosphorylated.
It is preferred that the biological sample comprises protein.
It is also preferred that a reduction in protein phosphorylation is detected.
Preferably, the mammal is a human.
It is preferred that the autoimmune disease is an HLA class II-linked disease.
In another preferred embodiment, the autoimmune disease is selected from the group provided above.
Another aspect of the present invention is a method of detecting autoimmune disease in a mammal, comprising providing a biological sample from a mammal and detecting NFxcexaB activity, wherein a reduction in NFxcexaB activity from a basal state is indicative of autoimmune disease.
As defined herein with regard to NFxcexaB activity, the term xe2x80x9creductionxe2x80x9d refers to a loss of the ability of NFxcexaB to direct the transcription of genes whose cis-regulatory sequences comprise an NFxcexaB recognition site, wherein such a site is normally bound and transcription of the gene activated by NFxcexaB. Preferably, such a reduction is in the range of 5-10% of the basal state level of activity, more preferably 25-50% and most preferably 70-100%.
Preferably, the biological sample comprises protein.
It is preferred that the biological sample comprises a nucleic acid.
As used herein, the term xe2x80x9cnucleic acidxe2x80x9d refers to a DNA molecule, such as genomic DNA or cDNA, and also to RNA. A nucleic acid may be double- or single-stranded, circular or linear and may be naturally-occurring, recombinant or synthetic (produced by either enzymatic or chemical means as a known in the art); if recombinant or synthetic, a nucleic acid molecule may comprise sequences which are known to occur naturally or which are novel.
It is preferred that a reduction in said NFxcexaB activity is detected.
As stated above, a reduction in in NFxcexaB activity may be determined either through its failure to direct the transcription of downstream genes, physical characteristics or DNA- or protein-binding activity in comparison to those of the basal state. NFxcexaB activity may be assayed either in vivo or in vitro using an NFxcexaB-dependent reporter gene expression construct and a substrate for enzymatic detection (such as chloramphenicol acetyl transferase or xcex2-galactosidase, depending on the specificity of the enzyme encoded by the reporter gene), wherein comparative quantitation of the product of the diagnostic enzymatic reaction (or, in the absence of a reaction substrate, the level of the reporter mRNA or its encoded protein) in biological samples derived from a test subject and a normal control indivicual allow for the assessment of NFxcexaB functional loss. Alternatively, immunological or other biochemical determination of whether or not IxcexaB has been cleaved from NFxcexaB may be made, as described above and in Example 2, below.
Preferably, the mammal is human.
It is preferred that the autoimmune disease is an HLA class II-linked disease.
In another preferred embodiment, the autoimmune disease is selected from the group that includes those diseases listed above.
The invention also provides a method of detecting autoimmune disease in a mammal, comprising providing a biological sample from a mammal and detecting cell survival or growth, wherein cell death prior to direct lymphocyte or antibody attack in a tissue that is a suspected target of an autoimmune disease is indicative of the autoimmune disease.
As used herein, the term xe2x80x9cgrowthxe2x80x9d refers to mitosis or differentiation (acquisition of cell surface marders or specialized functions, e.g. protein production, indicative of a mature cell type.
As used herein, the tern xe2x80x9ctissuexe2x80x9d refers to intact tissue or tissue fragments, such that the cells are sufficiently aggregated (associated) so as to form a cohesive mass. A tissue may comprise an entire organ (e.g. the pancreas, the thyroid, a muscle, or others) or other system (e.g. the lymphatic system) or a subset of the cells thereof; therefore, a tissue may comprise 0.1-10%, 20-50% or 50-100% of the organ or system (e.g. as is true of islets of the pancreas).
Examples of tissue types that are the targets of autoimmune disease include, but are not limited to, blood, lymph, the central nervous system (including brain or spinal cord gray or white matter), liver, kidney, spleen, heart muscle or blood vessels, cartilage, ligaments, tendons, lung, pancreas (in particular, pancreatic islets of Langerhans), lacrimal ducts, melanocytes, the adrenal cortex, skin, the intestinal tract (in particular, the luminal epithelium and the colon), ovary, testes, prostate, and regions such as joints, nerve/blood vessel junctions, salivary glands, bones, specific tendons or ligaments.
As used herein, the term xe2x80x9ccellsxe2x80x9d is defined as including dissociated cells, intact tissue or tissue fragments.
As used herein, the term xe2x80x9csuspected targetxe2x80x9d refers to a tissue that is damaged in the course of an autoimmune disease of which a mammal is believed to suffer or to be at risk of suffering.
It is contemplated that an individual is at risk of an autoimmune disease based either upon family history, the results of genetic testing, exposure (either after birth or in utero) to a substance such as is known to trigger autoimmune disease (see, below, the description of animal models of autoimmune disease); such an individual is xe2x80x9csuspected of sufferingxe2x80x9d (see below) or xe2x80x9csuspected of harboringxe2x80x9d an autoimmune disease or is said to have a xe2x80x9cpropensityxe2x80x9d for developing such a disease.
Preferably, the sample is obtained from the mammal at an early stage in the disease prior to or early in the formation of autoantibodies against the tissue.
As used herein, the term xe2x80x9cpriorxe2x80x9d may refer to a period of time immediately before autoantibodies first are or would expected to be formed in an individual with a propensity for autoimmune disease. xe2x80x9cPriorxe2x80x9d may be used to indicate a time weeks, months or years before the appearance of autoantibodies. It is contemplated that in an individual suspected of being at risk for an autoimmune disease, this may be as early as birth or even during the prenatal period.
As used herein, the term xe2x80x9cearlyxe2x80x9d refers to a stage of an autoimmune disease preceding complete target tissue destruction by the immune system.
Preferably, cell death is detected in a tissue that is a suspected target of autoimmune disease prior to the formation of autoantibodies.
It is preferred that the biological sample comprises cells of a tissue that is a suspected target of autoimmune disease.
It is additionally preferred that the mammal is a human.
Preferably, the autoimmune disease is an HLA class II-linked disease.
In another preferred embodiment, the autoimmune disease is selected from the group that includes those diseases listed above.
The invention also encompasses a method of treating an autoimmune disease in a mammal, comprising administering to a mammal suspected of suffering from an autoimmune disease an agent which restores protein ubiquitinating enzyme function in an amount and for a time sufficient to result in normal protein ubiquitination in the mammal.
As used herein, the term xe2x80x9cagentxe2x80x9d refers to a biochemical substance selected from the group that includes, but is not limited to, proteins, peptides or amino acids; nucleic acids such as DNA, such as full-length genes or fragments thereof derived from genomic, cDNA or artificial coding sequences, gene regulatory elements, RNA, including mRNA, tRNA, ribosomal RNA, ribozymes and antisense RNA, oligonucleotides and oligoribonucleotides, deoxyribonucleotides and ribonucleotides; carbohydrates; lipids; proteoglycans; such agents may be administered as isolated (purified) compounds or in crude mixtures, such as in a tissue, cell or cell lysate. Alternatively, xe2x80x9cagentxe2x80x9d may refer to an organic or inorganic chemical as is known in the art.
Methods of administering a therapeutic agent include, but are not limited to, topical application (e.g., for skin lesions), intravenous drip or injection, subcutaneous, intramuscular, intraperitoneal, intracranial and spinal injection, ingestion via the oral route, inhalation, trans-epithelial diffusion (such as via a drug-impregnated, adhesive patch) or by the use of an implantable, time-release drug delivery device, which may comprise a reservoir of exogenously-produced agent or may, instead, comprise cells that produce and secrete the therapeutic agent.
As used herein, the term xe2x80x9cubiquitinating enzyme functionxe2x80x9d refers to the covalent attachment of one or more ubiquitin molecules to a protein by members of the several classes of ubiquitinating enzymes, which include ubiquitin-activating enzymes (E1, which prime ubiquitin for attachment to a protein), ubiquitin-conjugating enzymes (E2, which bind primed ubiquitin for transfer to a target protein and ubiquitin ligases (E3, which catalyze the linkage of ubiquitin to specific sites on the target protein, which sites vary in number and type from protein to protein, as discussed above).
As used herein with regard to protein ubiquitination, the term xe2x80x9crestorexe2x80x9d refers to a return of the ubiquitination of at least one site which is normally ubiquitinated (that is, a site that is ubiquitinated in the basal state, as defined above) and, preferably all such sites, but is not ubiquitinated in the course of an autoimmune disease. Preferably, in the restoration of a normal level and pattern of ubiquitination, 50% of such sites are restored, more preferably, 60-85% and, most preferably, 90-100%. Such percentages include only the ubiquitination of sites that are normally ubiquitinated in the protein in question. In addition, an elevation of ubiquitination beyond 100% of normal values is not encompassed by this definition. It is contemplated that a restoration is sufficient to allow proper (i.e., that which is qualitatively comparable to that observed in the basal state) recognition and cleavage of the protein so ubiquitinated by the proteasome.
Preferably, the agent is selected from the group that consists of a protein and a nucleic acid that encodes that protein.
It is preferred that the protein is selected from the group that includes a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and ubiquitin-ligases (E3).
Examples of human homologues of the yeast ubiquitination enzymes include, but are not limited to UbcH5 (which functions as an E2) and the MDM2 oncoprotein, which acts as a ubiquitin ligase, or E3.
Preferably, the agent is a nucleic acid which encodes an antisense RNA or a ribozyme.
It is preferred that the mammal is a human.
It is additionally preferred that the autoimmune disease is an HLA class 11-linked disease.
In another preferred embodiment, the autoimmune disease is selected from the group that includes those diseases listed above.
Another aspect of the present invention is a method of treating an autoimmune disease in a mammal, comprising administering to a mammal suspected of suffering from an autoimmune disease an agent which restores NFxcexaB activity in an amount and for a time sufficient to result in normal NFxcexaB activity in the mammal.
As used herein, the term xe2x80x9cnormal NFxcexaB activityxe2x80x9d refers to a value that is at least 25% of the activity of one or more of NFxcexaB and its subunits p50, p105 and p65 observed in the basal state, as defined herein above, preferably in the range of 30-90% and most preferably in the range of 95-100%. xe2x80x9cNormal NFxcexaB activityxe2x80x9d may not exceed 100% of NFxcexaB basal state activity.
Preferably, the agent is selected from the group that consists of a protein and a nucleic acid that encodes that protein.
It is preferred that the protein is selected from the group that includes a mutant- or wild-type NFxcexaB p50, NFxcexaB p52, a competitor of IxcexaB that does not bind NFxcexaB p50 or NFxcexaB p65 (e.g., the IxcexaB mutant described in Ex. Jour. Biol. Chem., 1998, 273:2931, herein incorporated by reference), a mutant- or wild-type NFxcexaB p65, tumor necrosis factor-xcex1, E-selectin, I-cam, and V-cam, interleukin-2, interleukin-6, granulocyte colony-stimulating factor, interferon-xcex2, Lmp2, Lmp7, a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), a ubiquitin-ligase (E3), a ubiquitin deconjugating enzyme (UCH), a protein kinase, a proteasome subunit and an antibody directed against one of the 240 kD and 200 kD human erythrocyte proteasome inhibitors, OF-2 and IxcexaB.
In another preferred embodiment, the agent is selected from the group that consists of a ribozyme, an antisense RNA molecule, a DNA molecule that encodes a said ribozyme, and a DNA molecule that encodes a said antisense RNA molecule.
Preferably, the ribozyme or antisense RNA molecule is directed against one of the 240 kD and 200 kD human erythrocyte proteasome inhibitors, OF-2 and IxcexaB.
It is preferred that the mammal is a human.
It is additionally preferred that the autoimmune disease is an HLA class II-linked disease.
In another preferred embodiment, the autoimmune disease is selected from the group that includes those diseases listed above.
Another aspect of the present invention is a method of treating an autoimmune disease in a mammal, comprising administering to a mammal suspected of suffering from an autoimmune disease resulting from a reduction in the activity of NFxcexaB, DNA repair factor TFIIH, STAT transcription factor, ubiquitination, phosphorylation or the proteasome an agent which restores lymphocyte maturation in an amount and for a time sufficient to result in normal lymphocyte maturation in the mammal.
It is preferred that the agent is selected from the group that consists of a protein and a nucleic acid that encodes that protein.
It is additionally preferred that the protein is selected from the group that includes apolipoprotein B100, DNA repair factor TFIIH, STAT transcription factors a mutant- or wild-type NFxcexaB p50, a mutant- or wild-type NFxcexaB p65, tumor necrosis factor-xcex1, E-selectin, I-cam, and V-cam, interleukin-2, interleukin-6, a ubiquitin deconjugating enzyme (UCH), colony-stimulating factor, interferon-xcex2, Lmp2, Lmp7, a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (2), a ubiquitin-ligase (E), a protein kinase, a proteasome subunit and an antibody directed against one of the 240 kD) and 200 kD human erythrocyte proteasome inhibitors, OF-2 and IxcexaB.
Preferably, the agent is selected from the group that includes a ribozyme, an antisense RNA molecule, a DNA molecule that encodes a ribozyme and a DNA molecule that encodes an antisense RNA molecule.
It is preferred that the ribozyme or antisense RNA molecule is directed against one of the 240 kD and 200 kD human erythrocyte proteasome inhibitors, OF-2 and IxcexaB.
It is additionally preferred that the mammal is a human.
Preferably, the autoimmune disease is an HLA class II-linked disease,
In another preferred embodiment, the autoimmune disease is selected from the group that includes those diseases listed above.
A final aspect of the present invention is a method of treating an autoimmune disease in a mammal, comprising administering to a mammal suspected of suffering from an autoimmune disease resulting from a reduction in the activity of NFxcexaB, DNA repair factor TFIIH, STAT transcription factor, or the proteasome an agent which restores the cell cycle in an amount and for a time sufficient to result in normal survival of cells of a tissue that is susceptible to an autoimmune disease prior to the formation of autoantibodies, prior to cell death or prior to cellular attack against the cells in the
As defined herein, xe2x80x9cnormal survival of cellsxe2x80x9d is at least a 10% cell survival rate relative to that observed in the basal state. Preferably, xe2x80x9cnormal survival of cellsxe2x80x9d is in the range of 25-50% or even 75-100%; however, xe2x80x9cnormal survival of cellsxe2x80x9d does not encompass cell survival at a rate higher than 100% of that observed in the basal state. In other words, xe2x80x9cnormal survival of cellsxe2x80x9d does not refer to hyperproliferation of cells.
Preferably, the agent is selected from the group that includes a protein and a nucleic acid that encodes that protein.
It is preferred that the protein is selected from the group that includes a cyclin, a cyclin-dependent kinase, apolipoprotein B100, DNA repair factor TFIIH, STAT transcription factor, a mutant- or wild-type NFxcexaB p50, a mutant- or wild-type NFxcexaB p65, tumor necrosis factor-xcex1, E-selectin, I-cam, and V-cam, interleukin-2, interleukin-6, granulocyte colony-stimulating factor, interferon-xcex2, Lmp2, Lmp7, a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), a ubiquitin-ligase (E3), a ubiquitin deconjugating enzyme (UCH),a protein kinase, a proteasome subunit and an antibody directed against one of the 240 kD and 200 kD human erythrocyte proteasome inhibitors, OF-2 and IxcexaB.
It is additionally preferred that the agent is selected from the group that includes a ribozyme, an antisense RNA molecule, a DNA molecule that encodes a ribozyme and a DNA molecule that encodes an antisense RNA molecule.
Preferably, the ribozyme or antisense RNA molecule is directed against one of the 240 kD and 200 kD human erythrocyte proteasome inhibitors, OF-2 and IxcexaB.
It is preferred that the mammal is a human.
It is additionally preferred that the autoimmune disease is an HLA class II-linked disease.
In another preferred embodiment, the autoimmune disease is selected from the group that includes those diseases listed above.
A final aspect of the invention is a method for screening for a modulator of LMP2 function, comprising the steps of contacting an assay system with a candidate modulator of LMP2, wherein in the system, proteasome-mediated cleavage of a ubiquitinated protein occurs, and monitoring cleavage of the ubiquitinated protein, wherein a change in cleavage resulting from the contacting indicates that the candidate modulator is effective as a modulator of LMP2 function.
Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.